Cyclophosphamide decreases nitrotyrosine formation and inhibits nitric oxide production by alveolar macrophages in mycoplasmosis

Abstract

We previously reported that congenic C57BL/6 inducible nitric oxide synthase(-/-) (iNOS(-/-)) mice infected with Mycoplasma pulmonis developed higher bacterial numbers and lung lesion scores than C57BL/6 iNOS(+/+) controls but had similar lung nitrotyrosine levels. The present studies investigated the role of inflammatory cells in nitrotyrosine formation during mycoplasmal infection. iNOS(+/+) and iNOS(-/-) mice were injected with cyclophosphamide (CYP) and inoculated with 10(7) CFU of M. pulmonis. CYP pretreatment of M. pulmonis-infected iNOS(+/+) and iNOS(-/-) mice reduced polymorphonuclear cells (PMNs) within bronchoalveolar lavages (BALs) by 88 and 72%, respectively, and whole-lung myeloperoxidase levels by 80 and 78%, respectively, at 72 h postinfection but did not alter the number of alveolar macrophages (AMs) in BALs. CYP treatment also significantly decreased nitrate and nitrite (NOx) levels in BALs and plasma of infected iNOS(+/+) mice, whereas neither CYP nor mycoplasmal infection altered NOx in iNOS(-/-) mice. CYP reduced lung nitrotyrosine levels in both iNOS(+/+) and iNOS(-/-) mice to uninfected-control levels as shown by immunohistochemical staining and enzyme-linked immunosorbent assay and inhibited mycoplasmal killing by iNOS(+/+) mice in vivo. CYP inhibited the production of gamma interferon-inducible NOx by iNOS(+/+) AMs in vitro but did not alter the number of iNOS-positive AMs, as detected by immunocytochemistry. In addition, AMs from CYP-treated iNOS(+/+) mice had significantly decreased ability to kill mycoplasmas in vitro. These results demonstrate that reactive species generated by inflammatory cells as well as PMN myeloperoxidase are important contributors to nitrotyrosine formation during mycoplasmal infection and that treatment with CYP decreases NO* production by AMs and inhibits mycoplasmal killing.

FIG. 1

Effect of CYP on cell counts and MPO. B6 iNOS^−/− and control B6 iNOS^+/+ mice were injected with 200 mg of CYP/kg i.p. at time zero and with 100 mg/kg 72 h later. Control mice were injected with sterile saline. All mice were infected with 1.5 × 10⁷ CFU of M. pulmonis at the time of the second CYP injection and euthanized at 72 h p.i. for determination of PMN counts in BAL fluid and MPO levels in whole-lung homogenates. Results are means ± SE (n = 16 to 24 mice).

FIG. 2

Nitrotyrosine immunohistochemistry. Visualization of nitrotyrosine residues in the lungs of B6 iNOS^+/+ or B6 iNOS^−/− mice pretreated with CYP or saline and infected with 1.5 × 10⁷ CFU of M. pulmonis for 3 days is shown. (A) B6 iNOS^+/+ nitrotyrosine staining with saline pretreatment. (B) B6 iNOS^+/+ nitrotyrosine staining with CYP pretreatment. (C) B6 iNOS^+/+ nitrotyrosine staining of PMN-rich area (same block as panel A) in the presence of excess nitrotyrosine (10 mM). (D) B6 iNOS^−/− nitrotyrosine staining after saline pretreatment. (E) B6 iNOS^−/− nitrotyrosine staining after CYP pretreatment. (F) B6 iNOS^−/− nitrotyrosine staining of neutrophil-rich area (same block as panel D) in the presence of excess nitrotyrosine (10 mM). Pictures are representative (n ≥ 6 slides per group from separate mice).

FIG. 3

Quantitation of BAL nitrotyrosine by ELISA. B6 iNOS^−/− and control B6 iNOS^+/+ mice were pretreated with CYP or saline i.p. and infected intranasally with 1.5 × 10⁷ CFU of M. pulmonis. All mice were euthanized at 72 h p.i., and their lungs were lavaged with 2 ml of sterile saline. Cells were removed after centrifugation, and supernatants were tested for nitrotyrosine (NT) by ELISA using rabbit antinitrotyrosine antibody. ✻, significant difference between CYP- and saline-treated groups. Results are means ± SE (n = 9 to 16 mice).

FIG. 4

Effect of CYP on lung histopathology. Shown are hematoxylin- and eosin-stained lung sections from B6 iNOS^−/− mice treated with CYP or saline and infected for 72 h with 1.5 × 10⁷ CFU of M. pulmonis. (A) Large-airway, saline-treated; (B) large-airway, CYP-treated; (C) alveoli with many PMNs, saline treated; (D) alveoli with rare PMNs, CYP treated. Pictures are representative of sections made for lung lesion analysis.

FIG. 5

Effect of CYP on lung lesion indices. Hematoxylin- and eosin-stained lung sections from B6 iNOS^−/− and control B6 iNOS^+/+ mice treated with CYP or saline and infected for 72 h with 1.5 × 10⁷ CFU of M. pulmonis were coded randomly and scored subjectively on the basis of characteristic lesions for respiratory mycoplasmosis: (i) neutrophilic exudate in the airway lumen (exudate); (ii) hyperplasia-dysplasia of the airway epithelium (epithelial hyperplasia); (iii) peribronchiolar and perivascular lymphoid accumulation (lymphoid hyperplasia); (iv) inflammatory infiltration of alveoli (parenchymal lesions). Asterisk, significant difference between CYP- and saline-treated groups (P < 0.05); pound sign, significant difference between saline-treated B6 iNOS^−/− and control B6 iNOS^+/+ mice (P < 0.05). Results are means ± SE (n = 13 to 19 mice).

FIG. 6

Effect of CYP on mycoplasma killing in vivo. B6 iNOS^−/− and control B6 iNOS^+/+ mice were treated with 300 mg of total CYP/kg and infected with 1.5 × 10⁷ CFU of M. pulmonis. All mice were euthanized at 72 h p.i., and the mean numbers of CFU (total recoverable mycoplasmas) on whole-lung homogenates were determined. Asterisk, significant difference from all other treatment groups (P < 0.05). Results are means ± SE (n = 20 to 24 mice).

FIG. 7

Effect of CYP on plasma and BAL NO₃⁻ and NO₂⁻ levels. B6 iNOS^−/− and control B6 iNOS^+/+ mice were treated with CYP or saline i.p. and infected intranasally with 1.5 × 10⁷ CFU of M. pulmonis or sterile mycoplasma broth. All mice were euthanized at 72 h, plasma was collected, and their lungs were lavaged with 2 ml of sterile saline. (A) Plasma NO₂⁻ levels (n = 18 mice for all groups). (B) BAL NO₂⁻ levels (n = 20 or 21 mice). NO₂⁻ levels were determined using the Greiss reagent after conversion of NO₃⁻ to NO₂⁻ with E. coli reductase. Asterisk, significant difference from all other treatment groups (P < 0.05). Results are means ± SE.

FIG. 8

Effects of CYP on SP-A-mediated killing of M. pulmonis by AMs in vitro. B6 iNOS^+/+ mice were treated with CYP or saline, and AMs were collected by BAL. AMs were activated with 100 U of IFN-γ/ml and infected with 10¹⁰ CFU of M. pulmonis in the presence or absence of SP-A (25 μg/ml). AMs were incubated at 37°C for 6 h and ruptured by sonication, and the remaining CFU were determined by quantitative culture. Results are means ± SE, with 7 to 11 data points per group. Asterisk, significant difference between control and experimental group (P < 0.05).

FIG. 9

Effects of CYP on NO₃⁻ and NO₂⁻ production. B6 iNOS^+/+ mice were treated with CYP or saline, and AMs were collected by BAL. AMs (10⁵) were plated onto Lab-Tek chamber slides, activated with 100 U of IFN-γ/ml, and treated with SP-A (25 μg/ml) in the presence or absence of M. pulmonis (10¹⁰ CFU). AMs were incubated at 37°C for 6 h, and media were collected for NO₂⁻ and NO₃⁻ measurements. NO₂⁻ levels were determined using DAN after conversion of NO₃⁻ to NO₂⁻ with E. coli reductase. Results are means ± SE (n = 6 to 8). Asterisk, significant difference between CYP-treated group and saline-treated control group (P < 0.05).

FIG. 10

Effects of CYP on iNOS staining. B6 iNOS^+/+ mice were treated with CYP or saline, and AMs were collected by BAL. AMs were plated onto Lab-Tek chamber slides, activated with 100 U of IFN-γ/ml, and treated with SP-A (25 μg/ml) in the presence or absence of M. pulmonis (10¹⁰ CFU). AMs were incubated at 37°C for 6 h, and cells were fixed with paraformaldehyde for iNOS determination. (A) iNOS staining after saline pretreatment and treatment with SP-A and mycoplasmas. (B) iNOS staining after CYP pretreatment and treatment with SP-A and mycoplasmas. (C) iNOS staining with nonspecific mouse IgG as the primary antibody, after saline pretreatment and treatment with SP-A and mycoplasmas. Pictures are representative (n ≥ 6 slides).